Dominant Negative p63 Isoform Expression in Head and Neck Squamous Cell Carcinoma

Abstract

p63, a member of the p53 family of genes, is vital for normal epithelial development and may play a critical role in epithelial tumor formation. Although p63 has been identified in various head and neck malignancies, a detailed analysis of which of the six isoforms of the p63 gene is present in normal mucosa and head and neck malignancies has not yet been performed. The study analyzed p63 isoform expression on the RNA and protein level in normal, diseased, and malignant mucosa of the head and neck to examine the differential expression of p63 isoforms in head and neck tumors versus adjacent nonmalignant tissue and to identify the predominant p63 isoform expressed in head and neck squamous cell carcinoma (HNSCC). Three experiments were performed. In experiment 1, p63 expression was analyzed by immunohistochemical analysis in 36 HNSCC specimens and matched normal tissue control specimens harvested from the same patient. Western blot analysis was also performed on matched specimens to confirm the identity of the p63 isoforms that were found. In experiment 2, reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on matched normal and tumor specimens to analyze and quantitatively compare p63 isoform expression at the RNA level. In experiment 3, p63 expression was evaluated by immunohistochemical analysis in oral lichen planus, a benign mucosal lesion marked by hyperdifferentiation and apoptosis. Immunohistochemical analysis, RT-PCR, and Western blot analysis of p63 were performed on HNSCC specimens and matched normal tissue control specimens. p63 expression in oral lichen planus specimens was also examined by immunohistochemical analysis. In experiment 1, analysis of 36 HNSCC specimens from various head and neck subsites showed p63 expression in all tumors and matched normal tissue specimens (36 of 36). Western blot analyses indicated that dominant negative (DeltaN) isoform p63alpha (DeltaNp63alpha) is the major isoform expressed at the protein level in tumors and adjacent normal tissue. In experiment 2, RT-PCR analyses of 10 matched specimens confirmed that, although all three DeltaNp63 isoforms (DeltaNp63alpha, DeltaNp63beta, and DeltaNp63gamma) are expressed in normal and malignant mucosa of the head and neck, DeltaNp63alpha is the predominant transcript expressed. In experiment 3, immunohistochemical analysis of p63 in the pro-apoptotic condition of lichen planus indicated that p63 is underexpressed as compared with normal mucosal specimens. Although all three DeltaNp63 isoforms are present in HNSCC, DeltaNp63alpha protein is the predominant isoform expressed in these malignancies. DeltaNp63alpha is also overexpressed in tumors compared with matched normal tissue specimens and is underexpressed in the pro-apoptotic condition of lichen planus. These findings suggest that DeltaNp63alpha plays an anti-differentiation and anti-apoptotic role in the mucosal epithelium of the head and neck, possibly playing a pivotal role in the formation of HNSCC. Currently, DeltaNp63alpha is an attractive target for mechanistic study aimed at therapeutic intervention.